learning and activity after irradiation of the young mouse brain analyzed in adulthood using...

Learning and Activity after Irradiation of the Young Mouse Brain Analyzedin Adulthood Using Unbiased Monitoring in a Home Cage Environment

Niklas Karlsson,a,1 Marie Kalm,a,1 Marie K. L. Nilsson,b Carina Mallard,b Thomas Bjork-Erikssonc andKlas Blomgrena,d,2

a Center for Brain Repair and Rehabilitation, Institute of Neuroscience and Physiology, University of Gothenburg, SE 405 30, Sweden; b Institute ofNeuroscience and Physiology, University of Gothenburg, SE 405 30, Sweden; c Department of Oncology, Sahlgrenska University Hospital, SE 413

45 Gothenburg, Sweden; and d Department of Pediatric Oncology, The Queen Silvia Children’s Hospital, SE 416 85 Gothenburg, Sweden

Karlsson, N., Kalm, M., Nilsson, M. K. L., Mallard, C.,Bjork-Eriksson, T. and Blomgren, K. Learning and Activityafter Irradiation of the Young Mouse Brain Analyzed inAdulthood Using Unbiased Monitoring in a Home CageEnvironment. Radiat. Res. 175, 336–346 (2011).

Cranial radiotherapy during the treatment of pediatricmalignancies may cause adverse late effects. It is important tofind methods to assess the functional effects of ionizing radiationin animal models and to evaluate the possible amelioratingeffects of preventive or reparative treatment strategies. Weinvestigated the long-term effects of a single 8-Gy radiation doseto the brains of 14-day-old mice. Activity and learning wereevaluated in adulthood using open field and trace fearconditioning (TFC). These established methods were comparedwith the novel IntelliCage platform, which enables unbiasedanalysis of both activity and learning over time in a home cageenvironment. Neither activity nor learning was changed afterirradiation, as judged by the open field and TFC analyses. TheIntelliCage, however, revealed both altered activity and learningimpairment after irradiation. Place learning and reversallearning were both impaired in the IntelliCage 3 months afterirradiation. These results indicate that activity and learningshould be assessed using multiple methods and that unbiasedanalysis over time in a home cage environment may offeradvantages in the detection of subtle radiation-induced effects onthe young brain. g 2011 by Radiation Research Society

INTRODUCTION

Brain tumors constitute approximately one-third of allchildhood neoplasms, and survival rates after treatmentof primary or metastatic tumors located within or close tothe central nervous system (CNS) have increased over thelast decades (1). Despite improved techniques in neuro-surgery and advances in chemotherapy, radiation therapy

remains an essential treatment modality for malignantbrain tumors as well as for CNS involvement of leukemiaand lymphoma. However, radiation therapy is also one ofthe major causes of long-term complications seen insurvivors of pediatric brain tumors. Intellectual andmemory impairments as well as perturbed growth andpuberty are some of the late effects seen after radiationtherapy (2–6). These impairments have been shown to bemore severe in children younger than 3 years of age at thetime of radiation therapy (7–9).

Radiation-induced damage to the brain involves apop-tosis and loss of cells in the surrounding healthy braintissue and has been reported in the immature, juvenile andadult rat brain (10–15). A number of studies haveinvestigated the molecular mechanisms of injury to healthytissue, aiming to develop novel strategies to protect thebrain after irradiation. Behavioral changes after irradia-tion to the brain have previously been studied using, e.g.,the Morris water maze and open field (16, 17). To ourknowledge, there are no studies where irradiated animalshave been studied in their home cage environment, withminimal handling and disturbance, over longer periods. Itis reasonable to assume that testing in a social context overtime would reveal more subtle changes not apparent whenanimals are tested individually. The IntelliCage platformwas designed to minimize interaction between theexperimenter and the animals and is therefore aninteresting tool for the assessment of functional outcomein any brain injury paradigm. The aim of this study was toinvestigate the long-term effects of a single dose ofradiation to the young brain by using two establishedmethods, one for motor activity (open field) and one forlearning (trace fear conditioning), and then compare theseresults with those obtained using the IntelliCage system.

MATERIALS AND METHODS

Animals

C57BL/6 male mice were used (Charles River Laboratories,Sulzfeld, Germany). The animals were kept on a 12-h light cycle.

1 These authors contributed equally.2 Address for correspondence: Center for Brain Repair and

Rehabilitation, Institute of Neuroscience and Physiology, Universityof Gothenburg, Box 432, SE 405 30 Gothenburg, Sweden; e-mail:[email protected].

RADIATION RESEARCH 175, 336–346 (2011)0033-7587/11 $15.00g 2011 by Radiation Research Society.All rights of reproduction in any form reserved.DOI: 10.1667/RR2231.1

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Food and water were provided ad libitum. At the time of weaning, all

mice were anesthetized with isoflurane, and microtransponders were

injected subcutaneously (DATAMARS, PetLink, Youngstown, OH)

for identification purposes. After weaning, the animals were kept ingroups of up to 10 animals. All animal experiments were approved by

the local committee of the Swedish Animal Welfare Agency (46/2007

and 326/2009).

Irradiation

The irradiation procedure has been described previously (13, 18).On postnatal day 14, the pups were anesthetized with an intraper-

itoneal injection of 50 mg/kg tribromoethanol (Sigma, Stockholm,

Sweden) and placed in prone position on a polystyrene bed. The micewere irradiated using a linear accelerator (Varian Clinac 600 CD;

Radiation Oncology Systems LLC, San Diego, CA) with 4 MV

nominal photon energy. To obtain a uniform radiation dosethroughout the tissue, a 1-cm tissue-equivalent material was placed

on the head before irradiation. The whole brain was irradiated with a

2 3 2-cm field and a radiation source-to-skin distance of approxi-

mately 99.5 cm. The dose variation within the tissue was estimated to±5%. A single 8-Gy dose (2.3 Gy/min) was administered, and after

irradiation the animals were returned to their dams for recovery.

Control animals were anesthetized but not exposed to radiation. The8-Gy dose is approximately equivalent to 18 Gy when delivered in

repeated 2-Gy fractions using to the LQ model (19) and assuming an

a/b ratio of 3 for late effects in normal brain tissue. This represents a

clinically relevant dose, equivalent to the dose administered forexample in cases of childhood acute lymphoblastic leukemia with

CNS involvement. The doses used for malignant pediatric brain

tumors are often higher, up to 55 Gy.

Open Field

When the control and irradiated animals were 21 weeks old, theirmotor activity pattern was analyzed by video tracking. This

experimental procedure has been described previously by Nilsson et

al. (20). Briefly, the animals were individually introduced into anunfamiliar, open field arena and videotaped for 50 min. Four

indirectly illuminated arenas (l, w, h: 46 3 33 3 35 cm) were

videotaped simultaneously from above. The floors of the arenas were

covered with gray gravel that had been exposed earlier to other mice.

The recorded videos were analyzed with the video-tracking

software EthoVision 3.1 (Noldus Information Technologies bv,

Wageningen, the Netherlands). The program gives, for each samplingoccasion, the position of the mouse together with the animal’s body

area as seen from the overhead camera. The generated variables were

summarized into 10-min bins, and the video tracking was performed

at a sampling frequency of 12.5 Hz. Data were analyzed with respectto distance moved, stops, exploratory rearing, spatial variability in

the movement path, and percentage time spent in the middle of the

arena.

Trace Fear Conditioning

Nine and 15 weeks after irradiation, the animals were tested usingtrace fear conditioning. The animals were tested individually in an

observation chamber (Modified automatic reflex conditioner, Ugo

Basile, Comerio VA, Italy). The floor of each chamber consisted ofstainless steel rods. The rods were wired to a power source for the

delivery of a foot shock (0.5 mA). A speaker for delivering a tone was

mounted close to the box (80 dB, 670 Hz). On day 1 the mice wereplaced in the chamber and for 2 min the baseline freezing was

measured, followed by a tone for 30 s and a pause for 20 s, and then

the foot shock was delivered for 2 s. One day and 6 weeks after the

foot shock, the baseline freezing (2 min) and the freezing after thetone (2 min, this time without a foot shock) were measured. The

experiment was videotaped to facilitate analysis of the freezingbehavior.

IntelliCage

The IntelliCage platform has been described elsewhere (21–24).Briefly, each IntelliCage contains four learning and conditioningcorners (Fig. 1). Each conditioning corner is equipped with anantenna that registers the implanted microtransponders when themice enter the corner. In addition, each corner contains two waterbottles that can be accessed separately using doors. Sensors in eachcorner register nose pokes on the doors. Lick sensors register drinkingfrom the water bottles (Fig. 1B). Several IntelliCages can beconnected to a computer that registers events in the cages. Thesoftware can be programmed to act on specific events in the cages.For instance, when an animal performs a nose poke, the computercan open the door to allow the animal access to the correspondingwater bottle. In each corner it is also possible to activate an air valvefor administration of a negative reinforcement (air puffs) or to turnon up to six different LED lights of different colors (red, blue, greenor yellow) in response to an event (Fig. 1B).

The animals in this study were housed in the IntelliCages in groupsof up to 10 animals per cage and were 16 weeks old at the start of theIntelliCage experiment. Each experiment started with 5–6 days ofintroduction to the IntelliCages when the animals were acclimatizedto the cages, learning where to drink and to perform nose pokes togain access to the water bottles. To test the animals’ preference forsugar water, one water bottle in each corner was supplemented with0.1% fructose throughout the experiment. The introduction periodwas followed by a corner learning period where each animal wasrandomized to one corner (the most visited corner excluded); thiscorner was programmed as the correct corner while the three othercorners were programmed as incorrect (Fig. 1C). The mice were onlyallowed to drink from the water bottles (both plain water andfructose-supplemented water) in the correct corner, where nose pokeswould give them access to the water bottles. In incorrect corners, thedoors to the water bottles remained closed after nose pokes. After5 days, the animals were randomized to a new corner (the previouscorner excluded), and after another 5 days the animals wererandomized again to a new corner (the previous two cornersexcluded). The effects of negative reinforcement in the form of airpuffs and positive reinforcement in the form of LED lights wereevaluated in separate experiments. In two separate experiments, theair valve was activated when the mice made a nose poke in anonallocated corner or the green LED lights were turned on when themice entered a correct corner. Animals that failed to drink in theIntelliCages or were not registered at all (indicating misplaced,missing or malfunctioning microtransponders) were removed fromthe IntelliCages. Food was provided ad libitum during the experi-ments in the IntelliCage, and four red plastic houses were provided asshelters in the center of the cage (Fig. 1D).

In one experiment, SocialBoxes were connected to three Intelli-Cages in an attempt to assess social preference. SocialBox is an add-on to the IntelliCage system allowing for monitoring of the spatialand temporal distribution of mice and possibly individual socialpreference. Three smaller cages (26.7 3 20.7 3 14.0 cm) wereconnected to each of the larger IntelliCages through plastic cylindersequipped with antennas. The antennas register when a mouse leavesand re-enters the IntelliCage, allowing for registration of when andfor how long each mouse was present in a SocialBox together withother individual mice.

Data from the IntelliCages and the SocialBoxes were analyzedusing the IntelliCage software (IntelliCage Plus, 2.4, NewBehaviorAG, Zurich, Switzerland) together with Microsoft Excel 2007 andSPSS 17 (SPSS Inc., Chicago, IL). Only the active period (darkperiod) was analyzed, and visits lasting longer than 180 s, or where nonose poke was made, were removed from the analysis. The cutoff of

ALTERED COGNITION AND ACTIVITY AFTER IRRADIATION 337

180 s was chosen to remove all visits where the mice entered the

corners only to rest or sleep. The median visit duration was 18 s for

the control group and 19 s for the irradiated mice. Visits shorter than

3 min constituted 99.14% of all visits in the controls and 99.18% in

the irradiated mice; i.e., 0.86% and 0.82% were excluded in the

control and irradiated group, respectively. Animals that performed

six or fewer of two of the three measured parameters (visits, nose

pokes and licks) per day were excluded from the experiment.

Statistical Analysis

The trace fear conditioning data were analyzed using the Wilcoxon

signed rank and Mann-Whitney U tests. Time to first corner and lick

preference were analyzed using the Mann-Whitney U test. The

olfaction test and the exclusion rate from the IntelliCage when using

air puffs were analyzed using Fisher’s exact test. Preferences in the

SocialBox were analyzed using the Wilcoxon signed rank test. A

statistician evaluated the IntelliCage data and selected Generalized

Estimating Equations (GEE) to estimate and compare the average

responses of the populations for the different parameters measured in

the IntelliCage. GEE was used also for the open field data.

The IntelliCage experiments generate many quantities of interest.

From a statistical modeling perspective, there are three main types of

response variables: integer-valued variables (e.g., number of nose

pokes, number of visits in incorrect corner, etc.), binary variables

(quantified in terms of proportion of ‘‘success’’, e.g., proportion of

visits in incorrect corners), and continuous variables such as the visit

duration in incorrect corners. All quantities have been calculated

based on 12-h activity periods, so each experiment generates five

consecutive observation periods per animal for each responsevariable. The IntelliCage data can thus be thought of as longitudinaldata (repeated measurements over time) and require analysis methodsthat take the within-animal associations into account. Commonstatistical models for longitudinal data are Linear Mixed Models(LMM) and Generalized Estimating Equations (GEE). The repeated-measurement ANOVA is a special case of LMM. The LMMs assumea normally distributed response variable and are thus mainly suitablefor continuous responses. To analyze all types of responses within thesame model framework, we decided to use the GEE throughout. Wedid investigate the LMM approach as well, and for those responseswhere both models were applicable, the results based on an LMMagreed well with the corresponding GEE results.

There are different ways to account for the within-animalassociation in the GEE (as well as in the LMM). The three correlationstructures considered in the model selection analyses were theunstructured, which is the most general correlation model whereeach pair of measurements can have its own unique correlation, theauto-regression type of structure, where the pairwise correlationdecreases as the time between the two occasions increases, and theexchangeable structure, in which every pair of observations has aconstant correlation. However, the different covariance structuresturned out to have minimal effects on the model fits. Therefore, tokeep the number of estimated parameters to a minimum and therebyincrease the power of the tests, we decided to use the exchangeablecovariance structure in the final models.

The final model fitting procedure was performed as follows: Foreach response variable, a GEE model was set up with treatment andtime included both as main effects and as interaction. Time was

FIG. 1. Activity and learning were investigated using the IntelliCage platform. Panel A: A representativepicture of an IntelliCage. In panel B, a corner is shown. A schematic drawing of the IntelliCage is shown inpanel C. The green corner represents the allocated corner, and the three red corners represent the nonallocatedcorners. In panel D, a red plastic house is shown, four of which are placed in the middle of the IntelliCage, asindicated in panel C. The mice can seek shelter in the red houses or use them to reach the food grid above thehouses. 1 5 air puff valve, 2 5 presence detector, 3 5 three multicolor LEDs, 4 5 nose poke sensor, 5 5 a doorthat prevents access to one of the water bottles.

338 KARLSSON ET AL.

treated as a continuous covariate. Hence the model expression,

exemplified here by an integer-valued response, looked like

log m ijf g

� �~b0zb1treatizb2timejzb3treati � timej ,

where mij is the mean number of e.g. nose pokes in incorrect corners

for mice in treatment group i during activity period j (i 5 1,2, and j 5

1,..,5). The interaction term was then evaluated. A significant

interaction effect would mean that the development over time is

different depending on treatment. If the interaction showed no

significant effect, the interaction term was excluded and the model

was refitted. The effects of treatment and time were then considered

independent and were evaluated separately from each other in the

final results. For integer values, a Poisson-model was used, for ratios

a binominal model was used, and for other values a normal log model

was used. Differences between the groups were calculated from the

obtained b values. Statistical analysis was performed using SPSS 17.0

(SPSS, Chicago, IL) and significance was assumed when P , 0.05. All

data are shown as means ± SEM.

RESULTS

General Assessments

Thirteen of the animals (26%) were lost to follow-updue to death (2 mice; 4%), disease (2 mice; 4%), fighting (7mice; 14%) or microtransponder malfunction (2 mice;4%), and these mice were evenly distributed between thegroups. Establishing a social hierarchy occurs when theanimals reach sexual maturity, around 8 weeks of age, i.e.,long before the IntelliCage experiment. Very little fightingwas observed once the mice were in the IntelliCages, andonly one mouse was removed during the 5 weeks in theIntelliCages. No significant differences in body weightwere seen between the treatment groups at the time of theIntelliCage experiment. The mean body weight was 25.5 ±

0.4 g (n 5 22) for the control animals while the irradiatedanimals weighed 25.1 ± 0.3 g (n 5 20).

The number of licks from bottles with regular orsweetened water was monitored to assess fluid prefer-ence in the IntelliCage. No significant difference wasseen between controls and irradiated animals (P 5

0.341). The control mice performed 63 ± 8% of theirlicks on bottles with regular water; for irradiated micethis ratio was 49 ± 8%.

No social preference could be detected using theSocialBox add-on. Control mice spent 52 ± 2% of theirtime in the SocialBoxes together with another controlmouse and consequently 48 ± 2% with an irradiatedmouse (n.s.). Irradiated mice spent 49 ± 3% of their timetogether with a control mouse and consequently 51 ±

3% with another irradiated mouse (n.s.).

Activity1. Open field

Activity patterns in the open field revealed nosignificant differences in any of the measured variablesbetween control and irradiated animals. During the

50 min of the test, both groups moved 5% less and made6% fewer stops over time (Fig. 2).

2. IntelliCage

In the IntelliCage, the total number of visits and thetotal number of nose pokes in the four corners wererecorded as measurements of motor activity. In general,every time the mice were assigned a new corner, theirmotor activity increased; this was true for both controland irradiated animals. Comparing the total number ofnose pokes revealed a significant difference betweencontrol and irradiated mice over time (treatment 3 time,Fig. 3A). During the first corner period, the controls made

FIG. 2. Open field movement patterns were analyzed by usingvideo tracking. In panel A, distance moved is shown, divided into 10-min bins. In panel B, the data for how often the mice perform arearing are shown. In panel C, the average number of stops is shown.¤ 5 time, ¤¤¤P , 0.001 for time. Data are shown as means ± SEM. IR5 irradiated, n 5 12 per group.


10% fewer nose pokes per day, whereas the irradiatedanimals made 5% fewer nose pokes per day. By day 5during the first corner period, the difference between thecontrol and irradiated mice was 30%. During the secondcorner period, all animals displayed a 5% daily decrease innose pokes over time; a similar trend was seen in the thirdcorner period (Fig. 3A). There were significant differencesin the total number of visits in the control and irradiatedmice over time (treatment 3 time, Fig. 3B). In the firstcorner period, the controls made 7% fewer visits per day,whereas the irradiated animals made 3% fewer visits perday. In the second corner period, the control animalsdisplayed a small increase, whereas the irradiated animalsmade 3% fewer visits per day (Fig. 3B). In the third cornerperiod, both control and irradiated animals made 4%fewer visits per day over time (Fig. 3B).

The time it took until the mice had visited all fourcorners after being placed in the IntelliCage can also beused as a measure of motor and exploratory activity.There was no difference comparing the controls (2060 ±

484 s) with the irradiated mice (1738 ± 383 s).

Learning1. Trace fear conditioning

Both control animals and irradiated animals remem-bered that they had been subjected to a foot shock in the

observation chamber, because they displayed increasedfreezing behavior when they were placed in the chamberagain 1 day after the foot shock, indicating that theyreacted to the contextual part of the trace fearconditioning test, but there was no difference betweenthe groups in freezing behavior time (38 ± 5% comparedto 41 ± 5%, Fig. 4). Both groups increased their freezingbehavior even further after the tone stimulus, indicatingthat they also reacted to the cued stimulus (Day 2,Fig. 4), but no differences were seen between the groups(59 ± 6% compared to 57 ± 6%, Fig. 4, Day 2,) Sixweeks after the first test, both groups were tested againto assess memory retention. The context-induced freez-ing was very similar to the test 6 weeks earlier (43 ± 4%compared to 40 ± 3%). However, after the tonestimulus, neither of the groups increased their freezingbehavior, and no differences were seen between thegroups (46 ± 5% compared to 45 ± 4%, Fig. 4, 6 weeks).Thus no difference was seen between the control andirradiated mice at any of the times.

2. IntelliCage: Incorrect nose poke ratio

Place learning (first corner) and reversal learning(second and third corners) after irradiation wereinvestigated using the IntelliCage platform. The learningtask was more difficult for every corner period, since itrequired the mice first to learn where to drink and thenin the two subsequent corners to unlearn what theylearned previously and to find the new allocated corner.The incorrect nose poke ratio is the number of nosepokes in incorrect corners out of the total number ofnose pokes for a specific day. There was a significantdifference between the control and irradiated mice overthe 5 days of the first corner period (treatment 3 time).During the second and third corner periods, there weresignificant differences between the control and irradiat-ed animals (treatment and time separately, Fig. 5A).Irradiated animals displayed a 68% higher odds of

FIG. 3. The visit and nose poke activity was measured usingIntelliCage. On the x axis, the three corner changes are presented anddivided into the five different days (active periods) when a mouse wasallocated to the same corner. Panel A shows the average total numberof nose pokes made per day. Panel B shows the average total numberof visits made per day. * 5 time 3 treatment, ¤ 5 time, *P , 0.05,¤¤¤P , 0.001. Data shown are means ± SEM. IR 5 Irradiated. n 5

11–19.

FIG. 4. Contextual and cue-induced memory was investigatedusing a trace fear conditioning paradigm. The animals’ memory wastested 1 day and 6 weeks after the foot shock. Data shown are means± SEM. IR 5 irradiated, **P , 0.01, n 5 17–20.

340 KARLSSON ET AL.

making incorrect nose pokes during corner period twoand 112% during corner period three. In addition, bothcontrol and irradiated animals showed reduced incorrectnose poke ratios over time during corner periods two

(18% improved odds per day) and three (10% improvedodds per day).

3. IntelliCage: Nose pokes per incorrect visit

The number of nose pokes per incorrect visitrepresents the average number of times a mouseperformed a nose poke when it was in an incorrectcorner (Fig. 5B). A higher number of nose pokes perincorrect visit would reflect persistence in trying to openthe doors in incorrect corners, possibly indicatinglearning difficulties. The irradiated mice performed50% more nose pokes per incorrect visit compared withcontrol mice in the third corner period (Fig. 5B). Therewas a significant change over time during the first andsecond corner periods, but there was no differencebetween the groups. The change in nose pokes perincorrect visit was 6–8% per day (Fig. 5B), indicatinglearning.

4. IntelliCage: Incorrect visit ratio

The incorrect visit ratio is the number of times amouse enters a corner where they cannot drink dividedby the total number of corner visits. The animals showeda small but significant difference over time between thetreatment groups in corner period one (treatment 3

time). There was a trend for the irradiated animals tomake more incorrect visits in corner periods two andthree (P 5 0.06 and P 5 0.08, respectively). The groups’odds of making incorrect visits improved (decreased)10–17% per day (Fig. 5C).

5. IntelliCage: Time per incorrect visit

The time per incorrect visit represents how much time,on average, a mouse spent in an incorrect corner. Thecontrol animals spent significantly less time per visit inthe incorrect corners compared with the irradiated miceduring corner period three (Fig. 5D). The irradiatedanimals spent an average of 85% more time per visit inthe incorrect corners. Both control and irradiatedanimals showed a significant increase in time perincorrect visit over time (3–6%, Fig. 5D).

6. IntelliCage: Unlearning the previous corner

The number of nose pokes per previously correctcorner visit represents the average number of nose pokesperformed in the corner that was correct during theprevious corner period. There was a significant differ-ence between the groups over time during corner periodtwo (treatment 3 time, Fig. 6A). The control animalsmade significantly fewer attempts to drink in thepreviously correct corner, indicating that they hadlearned that they had to visit a new corner to be ableto drink. During the second corner period, the controlanimals made 15% fewer attempts per day, whereas the

FIG. 5. Learning was investigated using the IntelliCage system.On the x axis the three corner changes are presented and divided intothe five different days that a mouse was allocated to the same corner.Panel A: The incorrect nose poke ratio; panel B: the average numberof nose pokes each mouse made in an incorrect corner; panel C: theincorrect visit ratio; panel D; the average time that a mouse spent pervisit in an incorrect corner. * 5 time 3 treatment, ¤ 5 time, # 5

treatment, *, ¤, #P , 0.05, ¤¤, ##P , 0.01, ¤¤¤P , 0.001. Data shown aremeans ± SEM. IR 5 Irradiated. n 5 11–19.


irradiated animals made 2% fewer attempts per day.This resulted in a 94% difference between the controland irradiated group by day 5, indicating that theirradiated animals did not seem to understand the newtask. During corner period three, irradiated animalsmade 29% more attempts to drink in the previouslycorrect corner, and both groups improved 7% per day.

The nose poke ratio in the previously correct cornerrepresents the number of nose pokes in the previouslycorrect corner out of the total number of nose pokesmade that day. A significant difference between the twogroups over time was seen during corner period two(treatment 3 time, Fig. 6B). Control animals decreasedtheir odds of making nose pokes in the previouslycorrect corner by 28%, irradiated mice decreased theirodds by 16%. By day 5 the difference between the groupswas 141%, indicating that the irradiated animals hadproblems learning (unlearning the old correct corner).During corner period three, both groups decreased theirodds of making nose pokes in the previously correctcorner by 24% per day.

7. IntelliCage: Negative reinforcement

We studied learning in the IntelliCage in the absenceand in the presence of negative reinforcement. When amouse performed a nose poke in an incorrect corner, itwas punished with a short air puff. Exposing the animalsto air puffs in the incorrect corners led to unacceptably

high exclusion rates, 63% and 58% in the control andirradiated groups, respectively, compared to 0% withoutair puffs (P , 0.001). Exclusion was typically due to noor very low numbers of visits and attempts to drink.

8. IntelliCage: LED conditioning

In a separate experiment, a green LED light was litwhen animals entered the correct corner to facilitatelearning. Contrary to our expectations, using LED lightsdid not improve the reversal learning curves of the mice(Fig. 7).

DISCUSSION

We aimed to investigate the long-term effects onbehavior after irradiation of the young brain. We usedtwo established methods, open field and trace fearconditioning (TFC), which mainly assess motor activityand learning, respectively. We also used the relativelynovel IntelliCage platform, with which both motoractivity and learning can be analyzed continuously in ahome cage environment. In pediatric survivors of braintumors or other malignancies where cranial radiother-apy was part of the treatment, cognitive impairment is acommon late effect. Survival rates have increaseddramatically over the last decades, increasing theawareness of learning deficits and other late effects thatmay not be apparent until months or years after thetreatment (25). It is known that children treated withhigh doses of radiotherapy display deficits in intellectualfunction, academic achievement, memory, attention andprocessing speed (26). The difference between thepatients and their peers usually becomes more pro-nounced over the years to follow, as the survivorsdevelop at a slower rate. Observed declines in IQ aremost likely a result of failure to learn at a rate that isappropriate for the age of the child rather than from aloss of previously acquired knowledge (27). They

FIG. 6. Unlearning the previous corner was investigated using twodifferent parameters. Panel A: Nose pokes per previously correctcorner visit; panel B: the nose poke ratio in the previously correctcorner * 5 time 3 treatment, ¤ 5 time, # 5 treatment. Data shownare means ± SEM. IR 5 Irradiated, *, #P , 0.05, ¤¤P , 0.01, ¤¤¤P ,

0.001. n 5 17–19.

FIG. 7. Reversal learning (corner period three) in the IntelliCageswas investigated in the presence and absence of positive reinforcementin the form of a green LED, which was lit when the mice entered theallocated corners. * 5 time 3 treatment, ¤ 5 time, # 5 treatment.Data shown are means ± SEM. IR 5 irradiated, ¤, #P , 0.05, ¤¤P ,

0.01. n 5 8–18.

342 KARLSSON ET AL.

remember what they learned earlier but have difficultieslearning new things. This type of learning deficitindicates an impaired hippocampal memory system(28). Animal experiments have revealed that evenmoderate doses of ionizing radiation to the brain causedimmediate and extensive damage mainly to the neuro-genic regions of the brain, i.e. the hippocampus and thesubventricular zone (SVZ) (13, 29, 30), and that thiseffect was more pronounced in the young, still develop-ing brain (14). Furthermore, whereas neurogenesis in theSVZ partly recovered with time, the hippocampus didnot (31). According to some studies, impaired learning isassociated with decreased neurogenesis in the hippo-campus (24, 32–34). Given the vulnerability of the youngbrain in general and the hippocampus in particular toradiotherapy and the importance of the hippocampus inmemory and learning, it is important to find methods toassess not only the morphological effects but also thefunctional effects of radiation on the brains of rodentsand to evaluate the possible ameliorating effects ofpreventive or reparative treatment strategies.

We previously detected pronounced radiation-inducedchanges in motor activity 3 months after irradiationusing the open field test (16). In the current study wecould not detect any such changes using open field, eventhough the radiation dose used was higher than in theprevious study (8 Gy compared to 6 Gy). Thisdiscrepancy can be explained by the younger age ofthe mice at the time of irradiation in the previous study,postnatal day 9 compared to postnatal day 14. Atpostnatal day 9, the brain is growing rapidly, whereas atpostnatal day 14 the growth is leveling out and the brainis less sensitive to cranial irradiation. This is also true inhumans; the negative consequences of irradiation aremore severe in younger children (27). For this reason,cranial radiotherapy is rarely used in children youngerthan 4 years old. Investigating the degree of motoractivity using the IntelliCage platform is a novelapproach. When the animals were assigned a corner,the control and irradiated mice differed in theirexploratory behavior 3 months after irradiation. Duringthe first day of the first corner period, both groupsshowed similar nose poke frequencies, but by day 5 theirradiated mice displayed a higher nose poke frequency.This may reflect an initially equal level of purpose-oriented exploratory behavior, gradually shifting to alower level of activity in the control mice as they adaptedto their new conditions, whereas this change was lessclear in the irradiated mice. This is further supported bythe twice as rapidly decreasing visit frequency in thecontrol mice over the 5 days of the corner period. Ahigher activity in the irradiated animals could indicatehyperactivity was demonstrated earlier (16). The activityparameters recorded using the IntelliCage are notreadily comparable to the ones measured in the openfield. Thus using both methods will provide additional

information about the animals’ motor and exploratoryactivity.

Learning deficits are a major problem for childrenwho survive their brain tumors. Therefore, parametersreflecting memory and learning are relevant andimportant to study in preclinical settings. Learning afterirradiation has been studied using other tests, forinstance the Morris water maze (17). However, it haspreviously been shown that mice perform less well thanrats in the Morris water maze (37), suggesting that thistest is not as useful for mice. In addition, water mazetests can be affected by the stress associated with them(38), thereby involving not only the hippocampus butalso for example the amygdalae (28). One of theadvantages of the IntelliCage is that animal handlingby the experimenter can be virtually eliminated. Also,testing occurs in a social context, in a home cageenvironment over a long period.

Learning impairment after irradiation has beenstudied before but with tests involving excessivehandling of the animals in non-social environments(17, 39–41). We evaluated several different parametersto assess learning in the IntelliCage. Incorrect nose pokeratio and nose pokes per incorrect visit turned out to bevery useful. For example, the incorrect nose poke ratioclearly demonstrated that learning took place in all miceduring every corner period, as judged by the decreasingratio over the 5 days, followed by an increased ratioagain when the mice are assigned to a new corner.Furthermore, the incorrect nose poke ratio coulddiscriminate between control and irradiated mice, andthe difference was more apparent as the task grew moredifficult, i.e., as the allocated corner changed (Fig. 5A).To further refine the analysis, the number of nose pokesmade in the previously correct corner (Fig. 6) wasanalyzed and revealed that the irradiated mice hadgreater difficulties unlearning their old task and learninga new one. Incorrect visit ratio and time per incorrectvisit also proved to be useful parameters, but parametersbased on visits appeared to be less useful, presumablybecause the mice visit corners not only to drink but alsoto explore, hide or sleep. Nose pokes, on the other hand,appear to be more purpose-oriented and hence moreuseful. This was demonstrated by the lack of differencein incorrect visit ratio (Fig. 5C), whereas incorrect nosepoke ratio was significantly different during the sameperiod (Fig. 5A). Parameters based on visits would bemore useful if a negative stimulus could be applied,preventing visits to incorrect corners and hopefullypromoting visits to the correct corner. However, anegative stimulus in the form of a brief air puff was toostrong for most of the mice in our setup, yieldingunacceptably high exclusion rates. Air puffs caused 60%of the mice to avoid not only the incorrect corners butall corners, ultimately preventing them from drinking,and did not seem to improve learning among the animals


that were able to cope with the negative stimulus (datanot shown). Hence aversive stimuli like air puffs may becounterproductive in this context. Learning based on fearmay reflect a different neurobiological process, includinginvolvement of the amygdalae during negative reinforce-ment (42, 43), and it appears that the amygdalae are lessaffected by radiation than the hippocampi. Finding other,less stressful negative stimuli might therefore be useful. Insummary, investigating parameters based on nose pokessuch as ratio of incorrect nose pokes, nose pokes perincorrect corner, and nose pokes in previously correctcorner in the IntelliCages proved to be fruitful approach-es, because they revealed radiation-induced learningdeficits.

Trace fear conditioning (TFC) has been consideredappropriate to investigate hippocampal function (44).Given the vulnerability of the hippocampus to radiationand its importance in learning, we wanted to compareTFC with the IntelliCage platform. The TFC containsboth a context conditioning (being in the chamber thatpreviously gave a foot shock) and a cued conditioning(the response to a tone that was previously followed by afoot shock). We found that the TFC worked, becausethe animals responded with increased freezing behaviorwhen placed in the context and responded to the cue byfurther increasing their freezing behavior (Fig. 4). After6 weeks, the animals had lost the cue association andresponded only to the context. In neither case, however,did we find a difference in the freezing behavior betweenthe treatment groups. As mentioned in the previousparagraph, learning based on aversive stimuli activatessomewhat different regions of the brain, including theamygdalae (44). Fear conditioning may even have aninhibitory effect on memory formation (45). Although ithas previously been demonstrated that the radiationdose used in this study severely impairs cell division inthe hippocampus (13, 16), the damage does not seem tobe sufficient to cause differences in this established testof hippocampus/amygdalae function. Others have re-ported impairment in contextual fear conditioning usingslightly different protocols after irradiating (7.5 or 8 Gy)adult rats (46, 47). In both cases, radiation reducedfreezing time, and in one of the studies the animals werestill impaired when tested 48 h after the unconditionedstimulus (foot shock) (47). Saxe and colleagues (48)found, using cued fear conditioning, that the micelearned to associate the tone with the foot shock but sawno difference between irradiated and control animals.They did, however, find a difference in the context test.We cannot exclude that strain differences or repeatedtraining trials would yield differences after the tone. Insummary, the IntelliCage proved more useful in thisinjury paradigm than TFC in detecting learning deficits.

Radiotherapy-induced injury to the developing brainis aggravated over time (27). As the number ofchildhood cancer survivors has increased over the last

few decades, we have reached a greater understanding ofthe long-term cognitive deficits observed after suchtreatments. If the negative consequences of cranialradiotherapy could be ameliorated, the quality of lifeof the survivors and their families would increase. It isvital to find unbiased methods to measure cognitivedeficits in preclinical models to further our understand-ing of the mechanisms involved and to evaluate potentialtherapeutic interventions, including pharmacologicaland rehabilitation strategies. In this study, we showedthat a moderate radiation dose to the young rodentbrain caused injuries that were detectable in theIntelliCage, but not in open field or TFC, after severalmonths. These results indicate that activity and learningshould be assessed using multiple methods and that thenovel IntelliCage platform is a useful tool to detect moresubtle effects of radiation on the young brain.

ACKNOWLEDGMENTS

This work was supported by the Swedish Research Council(Vetenskapsradet), the Swedish Childhood Cancer Foundation(Barncancerfonden), the King Gustav V Jubilee Clinic CancerResearch Foundation (JK-fonden), Swedish governmental grants toscientists working in health care (ALF), the Frimurare BarnhusFoundation, the Wilhelm and Martina Lundgren Foundation, theGothenburg Medical Society, Edit Jacobson’s Foundation, theSahlgrenska Foundations and the Swedish Society of Medicine. Weare grateful for the skillful technical assistance of Rita Grander.Marita Olsson is greatly acknowledged for help with the statistics.

Received: April 9, 2010; accepted: October 12, 2010; published online:December 28, 2010

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